Nucleic acid amplification, and the polymerase chain reaction (PCR) in particular, has become an important research tool, with applications in cloning, analysis of genetic expression, DNA sequencing, genetic mapping, drug discovery, and the like (Gilliland etal, 1990; Bevan etal, 1992; Green etal, 1991). Descriptions of, and guidance for conducting, PCR is provided in extensive literature on the subject (Innis etal, 1989; McPherson etal, 1991; McPherson etal, 1995).
Automated instrumentation has been developed for carrying out nucleic acid amplifications, most commonly automated thermal cyclers for conducting PCR and the like. Important design goals fundamental to PCR instrument development have included fine temperature control, minimization of sample-to-sample variability in high-throughput, highly multiplexed-sample thermal cycling, automation of pre- and post-PCR processing steps, high-speed cycling, minimization of sample volumes, real time measurement of amplification products, minimization of cross-contamination or sample carryover, and the like. In particular, the design of instruments that permit PCR to be carried out and monitored in real time is highly desirable. Reaction chambers that remain closed during amplification and analysis are desirable for preventing cross-contamination (Higuchi etal, 1992; Higuchi etal, 1993; Holland etal, 1991).
Methods, reagents, and kits of reagents that facilitate multi-channel pipetting or robotic dispensing are desirable. A limited number of automated pipetting and dispensing steps will minimize errors and ambiguous results. Clearly, the successful realization of these design goals would be especially desirable in the analysis of diagnostic samples, where a high frequency of false positives and false negatives would severely reduce the value of the PCR-based procedure.
Fluorescence-based approaches to provide real time measurements of amplification products during a PCR (Holland etal, 1991) have either employed intercalating dyes (such as ethidium bromide) to indicate the amount of double stranded DNA present (Higuchi, 1992; Higuchi, 1993; Gelfand etal, 1993) or probes containing reporter-quencher pairs ("TaqMan.RTM.", exonuclease assay) that are cleaved during amplification to release a fluorescent signal that is proportional to the amount of double stranded DNA present (Livak 1996). The polymerase that conducts primer extension and amplifies the polynucleotide also possesses a 5'.fwdarw.3' exonuclease activity that serves to cleave the probe. In the exonuclease assay, a "reporter" dye and a "quencher" dye are attached to an oligonucleotide probe which is complementary to the target DNA. The dyes are selected and arranged to interact through a fluorescence resonance energy transfer (FRET) process (Clegg, R., 1992). The reporter is a luminescent compound that can be excited either by chemical reaction, producing chemiluminescence, or by light absorption, producing fluorescence (FIG. 1). The quencher can interact with the reporter to alter its light emission, usually resulting in the decreased emission efficiency of the reporter. This phenomenon is called quenching. The efficiency of quenching is a strong function of the distance between the reporter molecule and the quencher molecule. Thus, in a nucleic acid hybridization assay, detection of a hybridization event is accomplished by designing an energy transfer system in which the spacing between a reporter and a quencher is modulated as a result of the hybridization. Two examples of systems that perform the exonuclease assay and other quantitation, fluorescent-based arrays are the ABI PRISM.TM. 7700 and ABI PRISM.TM. 7200 Sequence Detection Systems (Perkin-Elmer).
The exonuclease assay of nucleic acid amplification employing reporter-quencher probes (Lee etal, 1993; Livak etal, 1995) gives direct detection of polymerase chain reaction (PCR) products with no downstream sample processing. The quencher is released from its close proximity to the reporter upon cleavage so that the signal from the reporter is no longer quenched. An increase in fluorescence occurs which correlates directly and proportionally with the increase in copies of the PCR product. By using real-time or end-point analysis, detection and quantitation of PCR products can be obtained by measuring the increase in fluorescence of cleaved, self-quenching fluorescent probes.
Release of fluorescence can be detected and measured by laser-induced fluorescence with an optical-fiber probe in a non-invasive, closed reaction chamber. The assay is conducted by a high-throughput, data sampling routine during the course of PCR thermal cycling (real-time analysis) or at the end of PCR (end-point analysis). Automated quantitation using PCR is highly desirable. Manual methods rely on end-point electrophoresis of an aliquot of the PCR and spectroscopic or densitometric quantitation. Real time monitoring of PCR permits far more accurate quantitation of starting target DNA concentrations in multiple-target amplifications than manual end-point methods. With calibrated, internal controls, the relative values of close concentrations can be resolved by factoring the history of the relative concentration values during the PCR. Real time monitoring of target with internal controls is desirable.
Internal and/or parallel tests that confirm conditions for amplification of the target are desirable as control tests. When used in a high-throughput format, such as 96 well, microtitre plate configurations, PCR often is plagued by false positives due to template contamination from adjacent wells, pipetting errors, or aerosol transmission. In addition, PCR suffers from false negative results when enzyme inhibitors are present in the target samples or when reagents are missing or degraded. Therefore, control amplification tests are desirable.
Positive amplification control tests give a detectable product derived from a component that is separate and distinct from the target. Detection of the positive control product indicates that amplification is viable and operative within the reaction chamber. Positive amplification control tests which give no detectable product from the control components, indicate conditions within the reaction chamber that do not allow amplification. Another desirable feature of amplification control is to "turn off" the positive control signal with the addition of a negative or "blocking" element to other reaction chambers during the assay, allowing the measurement of background.
Internal amplification controls are to be distinguished from "passive" internal reference molecules (Livak, Ser. No. 08/657,689) which provide for signal and detection corrections. Passive internal references, such as non-complementary, reporter-quencher molecules do not hybridize to target or other polynucleotides, are not consumed or act as substrates for enzymes, and do not undergo chemical or enzymatic reactions of any sort. Thus, passive internal references do not provide verification or indication of conditions for amplification, within the reaction chamber.
In addition to the above limitations and problems associated with signal and detection, a passive internal reference does not address the very common issues of; (i) contamination of target or other reagents with foreign DNA, (ii) inhibition of PCR, and (iii) confirming amplification efficiency within the reaction chamber. Reporter-quencher probe assays with internal fluorescence-generating controls are needed to provide accurate, precise, and sensitive measurements of changes in fluorescence that are attributable solely to formation of the amplification product.
Control amplification reactions are necessary for (i) normalization of quantitation results, (ii) detection of amplification inhibitors in the target and other reagents, and (iii) to establish background signal levels. A pervasive difficulty is keeping amplification of the control polynucleotide from interfering with target amplification or detection of the product. Internal control polynucleotides (ICP) undergo amplification within the same reaction chamber as the known or unknown target polynucleotide, imparting convenience in preparing samples and measuring results. The ICP may be endogenous, i.e. from the same source, genome, chromosome, gene, plasmid, or fragment as the target. Endogenous ICP are subject to amplification inhibitors and can therefore give a false negative signal. Endogenous ICP also may have priming sites for target primers and therefore give a false positive signal. In fact, endogenous ICP systems may share one or more primers with the target. Exhaustion of shared primers leads to inaccurate PCR quantitation and limited dynamic range. Another negative feature of endogenous ICP is the necessity to select, design, and purify ICP, ICP primers, and ICP self-quenching probe for each target to ensure compatibility and viable amplification. A universal exogenous ICP which is not derived from the same source, etc. as the target is therefore desirable, to avoid these disadvantages.
In addition to the positive control amplification, it is desirable to have separate, parallel negative control tests to establish a background signal level for quantitative systems such as the exonuclease assay. By "switching off" the control amplification, the fluorescence of the intact self-quenching probe of the internal control polynucleotide can be monitored and subtracted as a baseline value from samples undergoing normal amplification. Previous attempts at negative control background measurement have entailed; (i) deletion of essential components of amplification, such as magnesium, DNTP, and enzyme, or (ii) addition of amplification inhibitors such as sodium dodecyl sulfate (SDS) or EDTA. All of these attempts suffer from the disadvantage of changing, distorting, or obscuring fluorescent signal generation or detection.
Most techniques used for total RNA isolation yield RNA with significant amounts of genomic DNA contamination (Ambion). Reverse transcription, polymerase chain reaction (RT-PCR) is a popular method for analyzing low abundance mRNA from limiting amounts of tissue and the quantitative analysis of gene expression. Because the PCR step involves an exponential amplification, small tube-to-tube variations in amplification efficiency can translate into dramatic differences in the yield of final product and gross errors in estimation of initial abundance. A frequent cause of concern among investigators performing quantitative RT-PCR is inaccurate data caused by DNA contamination in RNA preparations. PCR cannot discriminate between cDNA targets synthesized by reverse transcription and genomic DNA contamination. Although DNA contamination is easily detected by performing a `no-RT` negative control, there is no satisfactory and comprehensive solution to contamination and inhibition which the present invention provides. The conventional methods of RT-PCR quantitative analysis (Wang etal, 1989; Tsai etal, 1996; Zimmermann etal, 1996) are all gel-based assays, which rely on imprecise and subjective visualization techniques.
In view of the limitations and deficiencies of conventional controls for the quantitation and detection of nucleic acid amplification products, it is of interest to develop non-gel based internal control methods that provide both negative and positive indications amplification. The invention described herein provides for such internal control reagents, kits, and methods.